Lecture 11

Question 1

The parent DNA double helix

Answer 1

• The two strands of the DNA double helix possess complementary sequence
• Each strand can serve as a template strand

Question 2

DNA replication

Answer 2

the process by which a copy of DNA is made

Question 3

Models of replication

Answer 3

• Semiconservative: each daughter molecule posses one parental strand
• Dispersive: daughter molecules have patches of old and new DNA
• Conservative: one daughter DNA is completely new DNA while parental strand remains intact

Question 4

DNA replication begin at:

Answer 4

• The origin which is rich in A-Ts
• DNA double helix opened with the aid of initiator proteins including helicase, single stranded binding (SSB) proteins, topoisomerases

Question 5

Replication origins

Answer 5

• Number of replication origins vary by species
• Example: Ecoli have 1, but humans have 103-104 per cell
• Having more can speed up DNA replication which is important for humans because they have much larger genomes
• E. coli can speed up DNA replication by initiating a new round of replication before completing the previous replication

Question 6

DNA replication requirements:

Answer 6

• DNA template
• Deoxyribonucleotide triphosphates
• A protein complex involving DNA polymerase enzyme
• RNA or DNA primer
• Mg 2+ ions (as a cofactor)

Question 7

In what direction does DNA replicate in

Answer 7

• DNA is replicated in a 5’ to 3’ direction
• Oncoming deoxyribonucleoside triphosphate provide the energy required for polymerization:
• The 3’ OH attacks the a-phosphate of the incoming dNTP. Pyrophosphate is released which is further hydrolyzed to two molecules of in organic phosphate

Question 8

Leading strand:

Answer 8

• One newly synthesized strand made continuously

Question 9

Lagging strand

Answer 9

• One strand is made discontinuously in short fragments known as Okazaki fragments

Question 10

What is DNA polymerase

Answer 10

• Enzyme responsible for catalyzing the addition of nucleotides to the 3’ end of a growing nucleic acid (there are several different DNA polymerase)
• Very accurate
• Requires an existing 3’ end to function (cannot begin a brand new strand)

Question 11

What are 4 contributing factors to polymerase accuracy

Answer 11

1. Hydrogen bonding of complementary base pairs
2. DNA polymerase monitors the base pairing before catalyzing the addition of the nucleotide
   * Active site only fits correct pairs
   * Correct base pairs form noncovalent bonds with DNA polymerase
3. Cell maintain roughly equal concentrations of deoxyribonucleoside triphosphates (dNTPs)
4. Proof reading: DNA polymerase can correct error using an 3’ to 5’ exonuclease

Question 12

Exonuclease:

Answer 12

Degrade nucleic acids from the end

Question 13

Endonuclease:

Answer 13

Cleave nucleic acids within a sequence

Question 14

Does DNA polymerase have separate sites for polymerization and error correcting?

Answer 14

• Yes there is a P and E site
• When an error occurs the incorrect nucleotide is pushed into the E site

Question 15

What is the function of primers:

Answer 15

• Dna polymerase cannot synthesize a new strand without a primer

Question 16

What is a primase:

Answer 16

• An RNA polymerase that creates RNA primers using the DNA template
• Primers will eventually be removed
• They do not have proof reading abilities

Question 17

Do we need a primer on both the lagging strand and the leading strand?

Answer 17

• Yes because the lagging strand requires several primers as polymerization continues and the leading strand requires a primer to start DNA replication at replication origin

Question 18

Lagging strand synthesis

Answer 18

• To create a new continuous strand from separate okazaki fragments the RNA primer is removed and replaced by DNA which is performed by “repair polymerase” which has proof reading function
• The nicks are sealed up by enzyme called DNA ligase
• 5’ to 3’ exonuclease activity is required

Question 19

What is the function of DNA ligase

Answer 19

• Seals gaps between two okazaki fragments during DNA replication
• Requires ATP
• Happens later on during DNA replication

Question 20

DNA replication requires coordinated effort of several proteins:

Answer 20

Helicases:
- unzips/ unwinds DNA double helix apart at the front of the replication fork
- requires ATP hydrolysis

Single stranded DNA binding proteins:
- bind to single stranded DNA preventing them from reforming base pairs

DNA topoisomerases:
- relieves tension that builds up in front of replication fork
- keeps DNA from super-coiling

Question 21

What are components of DNA polymerase

Answer 21

Sliding clamp:
- keeps DNA attached to the DNA

Clamp loader:
- locks new clamps onto DNA

Question 22

Primer problems:

Answer 22

• During replication the RNA primers are replaced with DNA, except for the primers used at the very 5’ end of the newly synthesized strands because DNA polymerase needs existing nucleotides to add incoming deoxyribonucleoside triphosphates (dNTPs)
• Therefore, this strand is vulnerable to degradation and
• DNA may get shorter with each round of replication

Question 23

Solution:

Answer 23

• The ends of our chromosomes contain repetitive, G and T-rich, non-coding sequences
• Telomeres attract enzymes known as telomerase which extend the length of telomeres using an internal RNA template
• The G’s are capable of forming a structure called G-quartet to protects the end of the chromosome (folds in on itself)

Question 24

Telomeres and aging:

Answer 24

• Eventually, enough DNA is lost and DNA containing important information is lost, so cells can no longer replicate

Question 25

HeLa cells:

Answer 25

- Telomerase activity is elevated in some tumour cells which unnaturally extends their life